摘要
以‘新梨7号’梨叶片为材料,探究影响原生质体分离的主要因素,制备高产优质的叶肉原生质体,并进行基因瞬时转化和表达。将继代30 d的组培苗嫩叶在最佳酶解液(1.0%纤维素酶,0.1%果胶酶,0.4%离析酶,0.5 mol·L^(-1)甘露醇,20 mmol·L^(-1)KCl,20 mmol·L^(-1)MES,10 mmol·L^(-1)CaCl_(2),1.0 g·L^(-1)BSA)中酶解12 h,可制备出产量为1.09×10^(6)protopl·mL^(-1)、活力为90%的原生质体。将1~2μg·mL^(-1)pROK2-GFP质粒DNA通过40%PEG-4000介导转化至上述原生质体中并培养12 h,可以观察到GFP绿色荧光信号,转化率可达40%。
High quality and quantity of protoplasts were succefully isolated from the mesophyll cells of leaves of tissue cultured‘Xinli 7'pear seedlings.This was achieved by optimizing the composition of the cell wall degrading enzymes and the reaction solution where the best results were obtained when the leaves of 30-days old tissue cultured pear seedlings were treated with 1.0%cellulase,0.1%pectinase and 0.4%macerozyme in a buffer containing 20 mmol·L^(-1)MES,0.5 mol·L^(-1)of D-mannitol,20 mmol·L^(-1)of potassium chloride,10 mmol·L^(-1)of calcium chloride and 1.0 g·L^(-1)of bovine serum albumin(pH 5.7).Approximately 1.09×10^(6)protopl·mL^(-1)were recovered with their viability being above 90%.To test their usefulness in genetic transformation in transient expression of proteins 1–2μg·m L^(-1)of pROK2-GFP plasmid DNA was transformed into the prepared protoplasts with 40%PEG-4000.After being cultured for 12 hours GFP was clearly observed in about 40%of the protoplasts,the transformation efficiency reached 40%.
作者
高成昱
王艺衡
靳江周
李涛
李金斗
周梦瑶
张海霞
马辉
张玉星
亓宝秀
许建锋
GAO Chengyu;WANG Yiheng;JIN Jiangzhou;LI Tao;LI Jindou;ZHOU Mengyao;ZHANG Haixia;MA Hui;ZHANG Yuxing;QI Baoxiu;XU Jjanfeng(College of Horticulure,Hebei Agricultural University,Baoding,Hebei 071051,China;Hebei Pear Technology and Innovation Center,Baoding,Hebei 071051,China;School of Pharmacy and Biomolecular Sciences,Liverpool John Moores University,Byrom Street,Liverpool L33AF,UK)
出处
《园艺学报》
CAS
CSCD
北大核心
2023年第5期1141-1150,共10页
Acta Horticulturae Sinica
基金
广东省重点领域研发计划项目(2020B0202010003)
国家重点研发计划项目(2019YFD1001404-5)
河北省重点研发计划项目(21326308D-1-4)
河北省现代农业产业技术体系梨创新团队项目(HBCT2018100202)
大学生创新创业项目(201810086040)。
关键词
梨
叶片
原生质体制备
瞬时转化
pear
leaf
protoplast isolation
protoplast transient transformation
